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Mechanism of eIF6 release from the nascent 60S ribosomal subunit

Nature Structural & Molecular Biology volume 22, pages 914919 (2015) | Download Citation

Abstract

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS–EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.

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Acknowledgements

We thank A. Johnson (University of Texas) and A. Newman (Medical Research Council Laboratory of Molecular Biology) for yeast strains; B. Trumpower (Dartmouth Medical School) for providing uL16 antiserum; S. Chen, C. Savva, S. De Carlo, S. Welsch, F. De Haas, M. Vos and K. Sader for technical support with cryo-EM; G. McMullan for help with movie data acquisition; T. Darling and J. Grimmett for help with computing; A. Brown for help with refinement; and S. Scheres for discussion. This work was supported by a Federation of European Biochemical Societies Long Term Fellowship (to F.W.), the Specialist Programme from Bloodwise (12048 to A.J.W.), the UK Medical Research Council (MRC) (MC_U105161083 to A.J.W. and U105115237 to R.R.K.), a Wellcome Trust strategic award to the Cambridge Institute for Medical Research (100140), a core support grant from the Wellcome Trust and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, the Tesni Parry Trust (to A.J.W.), Ted's Gang (to A.J.W.) and the Cambridge National Institute for Health Research Biomedical Research Centre.

Author information

Affiliations

  1. Cambridge Institute for Medical Research, Cambridge, UK.

    • Félix Weis
    • , Christine Hilcenko
    •  & Alan J Warren
  2. Medical Research Council Laboratory of Molecular Biology, University of Cambridge Research Unit, Cambridge, UK.

    • Félix Weis
    • , Mark Churcher
    • , Li Jin
    • , Christine Hilcenko
    •  & Alan J Warren
  3. Department of Haematology, University of Cambridge, Cambridge, UK.

    • Félix Weis
    • , Mark Churcher
    • , Li Jin
    • , Christine Hilcenko
    •  & Alan J Warren
  4. Wellcome Trust–Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.

    • Félix Weis
    • , Mark Churcher
    • , Li Jin
    • , Christine Hilcenko
    •  & Alan J Warren
  5. Université de Rennes 1, Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 6290, Institut de Génétique et Développement de Rennes, Rennes, France.

    • Emmanuel Giudice
  6. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

    • Li Jin
    • , David Traynor
    •  & Robert R Kay
  7. Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge, UK.

    • Chi C Wong

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Contributions

F.W. performed sample preparation, EM data collection, image processing and model refinement. E.G. performed model building and fitting; M.C., L.J. and A.J.W. performed genetic and biochemical experiments; C.H. performed protein expression and purification; C.C.W. generated mutant Dictyostelium strains with advice from D.T. and R.R.K.; and D.T. and F.W. cultured Dictyostelium cells. F.W. and A.J.W. designed experiments and wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alan J Warren.

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    Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Tables 1–6

  2. 2.

    Supplementary Data Set 1

    Uncropped blots related to Fig. 4

Videos

  1. 1.

    EFL1 conformational switch

  2. 2.

    SBDS conformational switch

  3. 3.

    Proposed mechanism of eIF6 release

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DOI

https://doi.org/10.1038/nsmb.3112

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